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Dec 18, 2015 - This is the first series of analogues of the novel selective SETD8 ... The emerging role of lysine methyltransferase SETD8 in human dis...
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Nahuoic Acids B−E, Polyhydroxy Polyketides from the MarineDerived Streptomyces sp. SCSGAA 0027 Xu-Hua Nong, Xiao-Yong Zhang, Xin-Ya Xu, Jie Wang, and Shu-Hua Qi* Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301 Guangdong, People’s Republic of China S Supporting Information *

ABSTRACT: Four new polyol polyketides containing a decalin ring, nahuoic acids B−E (1−4), together with a known analogue, nahuoic acid A (5), possessing an unprecedented carbon skeleton, were isolated from a culture broth of the marine-derived Streptomyces sp. SCSGAA 0027. Their structures were determined by detailed analysis of spectroscopic data and chemical transformations including acetonide formation and Mosher’s ester method. Compounds 1−5 showed weak antibiofilm activity against Shewanella onedensis MR-1 biofilm. This is the first series of analogues of the novel selective SETD8 inhibitor nahuoic acid A.

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spectroscopic analysis and chemical derivatization, including bis-acetonide formation, Mosher’s ester method, and calculated ECD spectra. Herein compound 5 was unambiguously identified as nahuoic acid A according to their matching NMR data and the same direction of specific rotation [[α]20D −1.6 (c 1.2, MeOH)] for 5 as that of nahuoic acid A [[α]25D −5.6 (c 3.2, MeOH)].5 Moreover, the experimental ECD curve for 5 was in accordance with that of nahuoic acid A, which further supported their identical absolute configuration. The structures of new compounds 1−4 were determined by parallel approaches used for nahuoic acid A (5), and the details of the structure elucidation are described below. Nahuoic acid B (1) had the molecular formula C30H50O8 as determined by HRESIMS. The 1H NMR spectrum (Table 1) displayed obvious signals for four secondary methyl groups (δH 0.87, 0.85, 0.78, 0.77), four tertiary methyl groups (δH 1.59, 1.47, 1.40, 1.15), and three olefinic protons (δH 6.43, 5.14, 4.99). The 13C NMR (Table 2) showed signals of a carboxyl group (δC 169.1), three olefinic methines (δC 148.8, 132.7, 125.3), 12 methines (δC 78.6, 75.6, 74.5, 70.1, 68.3, 67.1, 56.2, 50.6, 39.5, 36.2, 30.6, 30.3) including six carbinol methines, two methylenes (δC 41.6, 36.8), and eight methyl groups (δC 27.5, 21.4, 19.4, 18.4, 17.7, 12.9, 12.1, 7.0). These data showed great similarity to those of nahuoic acid A (5),5 which suggested that they had the same skeleton. Comparison of the NMR data of 1 and 5 displayed that the only obvious difference between them was the additional appearance of one oxymethine (δH 3.76, δC 74.5) and the absence of one shielded methylene in 1. The additional oxymethine was assigned to be C-8 on the basis of the COSY spectrum, showing correlations of H-8 (δH 3.76) with H-7 (δH 3.56)/H-9 (δH 3.42) (Figure 1). Further detailed

arine actinomycetes are distinguished as an important resource for drug development. Many natural products have been isolated from marine actinomycetes over the past decades, including alkaloids, peptides, macrolides, pyrones, and glycosides, which show antimicrobial, antioxidant, anticancer, and cytotoxic activities.1−3 Although numerous structure classes have been obtained from marine-derived actinomycetes, compounds with a decalin skeleton have been reported only a few times.4 Natural products containing decalin scaffolds represent a family of chemical entities correlated with highly multifunctionalized or architecturally complex groups, exhibiting diverse and remarkable biological properties such as antifungal, antibacterial, anticancer, and immunosuppressive activities. The decalin skeleton may arise from an enzymatic intramolecular Diels−Alder (IMDA) cycloaddition.4 In our investigations of the metabolic profiles of marine actinomycetes, five polyol polyketides containing a decalin ring, including four new nahuoic acids B−E (1−4) and the known analogue nahuoic acid A (5),5 were obtained from a culture broth of the marine-derived Streptomyces sp. SCSGAA 0027. Nahuoic acid A, containing an unprecedented carbon skeleton, was the first natural product inhibiting the SETD8 lysine methyltransferase and the first selective SETD8 inhibitor known to be a competitive inhibitor of (S)-adenosylmethionine binding. 5 Herein, we describe the isolation, structure elucidation, and bioactivities of 1−4.

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RESULTS AND DISCUSSION The strain Streptomyces sp. SCSGAA 0027 was cultured in liquid media for 5 days; then the broth was extracted with EtOAc to afford an organic extract. The extract was subjected to repeated column chromatography, including HPLC with an ODS column, to give new nahuoic acids B−E (1−4) and the known analogue nahuoic acid A (5).5 As reported, the complete structure of nahuoic acid A was established by © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 7, 2015

A

DOI: 10.1021/acs.jnatprod.5b00805 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Data for Compounds 1−4 in DMSO-d6 (δ in ppm, J in Hz, 500 MHz) no. 3 4

3

4

no.

1

6.39, d (11.0)

6.41, d (10.0)

3.59a

2.04, m

3.59 (ddd, 8.5,10.0,11.0) 1.40, dd (2.5, 10.0) 2.03a

3.55, td (8.5, 10.0) 1.54, dd (3.0, 8.5) 1.91, m

1.12, m 1.47a

1.45a 1.12, m

3.53, dt (1.0, 10.5)

1.82, tt (3.5, 13.5) 1.64a 3.36a 5.16, s 2.26, d (9.0) 4.99, br s 2.10, m 1.94, m 3.62a 1.47a 1.22, m 3.79, br d (6.5) 1.42a 3.15, ddd (4.0, 4.5, 7.0)

1.81, m

1.88, m

1.67a 3.36a 5.16, s 2.25, d (8.5) 4.99, br s 2.05a 1.95, m 3.63a 1.31, m

1.63a 3.48, m 5.16, s 2.24, d (8.5) 4.99, br s 2.05, m 1.95a 3.63, m 1.31, m

3.78a 1.43a 3.74a

3.80, m 1.45, t (6.5) 3.74, m

1.66a 0.85, d (6.5)

1.51a 3.16, m

1.59, s 0.87, d (8.5) 1.15, s 1.47, s 1.40, s 0.77, d (7.5) 0.78, d (8.0)

1.60, 0.82, 1.00, 1.49, 1.45, 0.77, 0.78,

169.1, 124.3, 148.8, 36.2, 50.6, 30.6, 68.3, 74.5, 75.6, 40.5, 132.7, 132.2, 56.2, 135.1, 125.3, 36.8, 67.1, 41.6, 70.1, 39.5, 78.6, 30.3, 18.4, 12.9, 17.7, 27.5, 21.4, 12.1, 7.0, 19.4,

11.78, br s 4.02, d (7.5) 4.26, d (5.0) 4.48, d (2.5) 4.30, d (7.0) 4.45, d (3.0) 4.31, d (4.5)

11.78, br s

1.64a 0.81, d (7.0) 1.60, s 0.82, d (7.5) 1.00, s 1.48, s 1.44, s 0.79, d (6.5) 0.86, d (6.5) 11.83, br s

1.51a 3.17, dd (4.0, 7.0) 1.67, m 0.86, d (7.0) 1.60, s 0.88, d (7.5) 1.02, s 1.48, s 1.41, s 0.78, d (7.0) 0.80, d (7.0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

a

1.49

6

2.37, ddq (3.5, 7.5, 11.0) 3.56, ddd (5.0, 7.5, 11.0) 3.76, dd (2.5, 5.0)

8

9 11 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 COOH 7-OH 8-OH 9-OH 17-OH 19-OH 21-OH 23-OH a

2 6.39, d (11.0)

1 6.43, d (11.0) 3.40a

5

7

Table 2. 13C NMR Data for Compounds 1−4 in DMSO-d6 (δ in ppm, 125 MHz)

3.42, br s 5.14, s 2.24, d (9.5) 4.99, br s 2.08, m 1.93, m 3.60, m 1.44a 1.23, m 3.79, m 1.41a 3.14, ddd (4.0, 4.5, 7.0) 1.67, m 0.85, d (6.5)

1.41

a

s d (7.5) s s s d (6.5) d (7.0)

4.18, br s 4.31, br d (4.5) 4.40, br d (4.5) 4.53, br d (3.0) 4.34, br d (4.0)

169.2, 125.2, 149.2, 35.2, 48.6, 31.2, 24.4, 30.0, 70.4, 41.2, 133.0, 132.7, 56.3, 135.1, 125.3, 36.9, 67.2, 41.6, 70.2, 39.7, 78.7, 30.4, 18.5, 12.9, 22.2, 27.4, 21.6, 12.1, 7.1, 19.4,

3 C C CH CH CH CH CH2 CH2 CH C CH C CH C CH CH2 CH CH2 CH CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

169.1, 124.3, 149.2, 34.9, 48.5, 31.2, 24.4, 30.0, 70.3, 41.1, 133.2, 132.0, 56.4, 135.2, 125.3, 36.8, 66.8, 43.9, 66.0, 42.3, 72.0, 38.6, 78.2, 30.3, 19.5, 12.9, 22.3, 27.3, 21.6, 12.0, 7.1, 18.3,

4 C C CH CH CH CH CH2 CH2 CH C CH C CH C CH CH2 CH CH2 CH CH2 CH CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

169.1, 124.9, 149.1, 35.9, 50.1, 38.3, 66.3, 39.9, 72.8, 41.1, 132.6, 133.0, 57.1, 135.6, 125.4, 36.7, 66.9, 43.8, 66.1, 42.3, 72.1, 38.7, 78.2, 30.3, 18.0, 12.9, 18.3, 27.6, 21.5, 12.2, 7.0, 19.4,

C C CH CH CH CH CH CH2 CH C CH C CH C CH CH2 CH CH2 CH CH2 CH CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

carbon chemical shift value of C-29 (δC 7.0) matched the typical value in Kishi’s Universal NMR Database for the methyl carbon in a 2-methyl-1,3-anti diol relationship (δC 7.3 in DMSO-d6). The result was further confirmed by derivatization of 1 to an acetone ketal. Treatment of 1 with 2,2dimethoxypropane and pyridinium p-toluenesulfonate (PPTS) in MeOH yielded an assumed bis-acetonide product. However, the reaction product was not stable and converted back to the original substrate of 1 immediately after preparation by HPLC on a C18 column. Thus, we modified the chemical reaction method. First, 1 was treated with 1% sulfuric acid in MeOH and incubated for 5 h at 70 °C to obtain a residue without purification. Subsequently, the residue was derivatized by the previous acetonide approach, affording the major bis-acetonide ketal 1a (Scheme 1). The 1D and 2D NMR spectroscopic data of 1a were similar to those of 1, and the main differences between them were the addition of the four sp3 methyl groups C-32 (δC 30.0), C-33 (δC 19.8), C-35 (δC 28.2), and C-36 (δC 25.8), the three sp3 oxygenated nonprotonated carbons C-12 (δC 80.7), C-31 (δC 98.9), and C-34 (δC 107.6), and the sp3 methylene C-11 (δC 53.9), and the absence of olefinic sp2 carbons C-11 (δC 132.7, CH) and C-12 (δC 132.2, C) (see the Supporting Information). These data indicated that two acetonides had formed and that an unexpected hydration of a double bond also occurred in 1a. Furthermore, the observed HMBC correlations from H3-32 (δH 1.37) to C-31/C-33, from

4.25−4.32b 4.27−4.31b 4.27−4.31b 4.27−4.31b 4.15,b d (2.5)

2 C C CH CH CH CH CH CH CH C CH C CH C CH CH2 CH CH2 CH CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

4.20,b, d (5.5) 4.25−4.32b 4.25−4.32b 4.54,b br s 4.25−4.32b

Overlapped signals. bNot determined.

analysis of the HSQC, HMBC, and COSY spectra of 1 (Figure 1) determined the planar structure of 1 as shown. The configuration establishment for 1 was segregated into two distinct parts of the decalin ring and the polyol side-chain segment. Initially, the relative configuration of the C-17/C-19/ C-20/C-21 cluster was deduced as anti/syn/syn by Kishi’s Universal NMR Database6,7 for 1,3-diol and 2-methyl-1,3-diol moieties (Figure 2). The observed carbon chemical shift value of C-17 (δC 67.1) matched the typical value in Kishi’s Universal NMR Database for an oxymethine carbon in a 1,3-anti diol relationship (δC 66.6 in DMSO-d6). Similarly, the observed B

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Figure 1. Key HMBC (↷) and COSY () correlations of 1 and 3.

Figure 2. Kishi NMR data sets (A−C) for elucidation of the relative configuration of 1,3,5-triols, 1,3-diols, and 2-methyl-1,3-diols in compounds 1− 4. ΔδC values between 1−4 and the model system are shown in brackets.

at δC 19.8 (C-33) and 30.0 (C-32), indicating the sixmembered 1,3-dioxane ring was in the chair conformation and the relative configuration of the C-19/C-20/C-21 cluster was syn/syn, consistent with the speculation from Kishi’s Universal NMR Database.8 As a proof of the deduced configuration from Kishi’s Universal NMR Database and 13C acetonide analysis, strong 1,3-diaxial NOE correlations between the axial geminal methyl (δH 1.42, δC 19.8) and H-19 (δH 4.15)/H-21 (δH 3.33) in the NOESY spectrum of 1a (Figure 3) illustrated a preferable chair conformation of the six-membered ring, where the hydroxy groups at C-19 (δC 70.3) and C-21 (δC 79.6) were both in

H3-33 (δH 1.42) to C-31/C-32, from H3-35 (δH 1.45) to C-34/ C-36, and from H3-36 (δH 1.35) to C-34/C-35 confirmed the formation of two acetonides. Additionally, the NOESY spectrum showing correlations from H3-33 (δH 1.42) to H-19 (δH 4.15)/H-21 (δH 3.33) and from H3-36 (δH 1.35) to H-7 (δH 4.13)/H-8 (δH 4.44) proved that the two acetonides occurred at C-7/C-8 and C-19/C-21, respectively. The observed HMBC correlations from H3-27 (δH 1.16) to C-11 (δC 53.9, CH2)/C-12 (δC 80.7, C)/C-13 (δC 63.0, CH) verified an acid-catalyzed hydration occurred at the C-11/C-12 double bond. Therefore, the planar structure of 1a was defined. The carbon chemical shifts of the acetonide methyl groups appeared C

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Scheme 1. Derivatization of Nahuoic Acids B and C (1 and 2)a

Reagents and conditions: (a) 1% sulfuric acid in MeOH, 70 °C; (b) 2,2-dimethoxypropane, MeOH, PPTS, rt; (c) pyridine-d5, dimethylaminopyridine and (R)-MTPACl/(S)-MTPACl, rt.

a

Figure 3. Key NOESY correlations for compounds 1a and 2.

in accordance with the above assignment. Additionally, the 3.0 Hz coupling value between H-17 (δ 3.74) and H-18a required a dihedral angle of near 60°. On the basis of the above data, the relative configuration of the polyol chain was determined. For determining the relative configuration of the decalin ring system of 1, a NOESY experiment was performed. Unfortunately, some key cross-peaks in the NOESY spectrum of 1 were obscured by noise. So, the relative configuration of the decalin ring unit of 1 was determined by unambiguous assignment of

equatorial positions. The NOE cross-peaks observed between H-20 (δH 1.36) and H-19/H-21 suggested that H-20 was also equatorial (Figure 3), which was confirmed by the combination of small vicinal coupling constants of J19−20 (2.5 Hz) and J20−21 (2.0 Hz).5 Moreover, observation of coupling constants between H-18a (δH 1.76) and H-19 (J18a‑19 = 11.5 Hz) and between H-21 and H-22 (δH 1.65) (J21−22 = 10.0 Hz) demonstrated these protons were anti. NOE correlations observed from H3-29 (δH 0.83) to H-18a and H-22 were also D

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Figure 4. ΔδS−R values for MTPA esters of 1a and 2a in CDCl3.

determined by the NOESY spectrum and coupling constants. The H-8ax resonance at δH 1.82 appeared as a triplet of triplets with the large coupling of 13.5 Hz, indicating the six-membered ring of C-5 to C-10 was in a chair conformation with the axial proton H-8ax. In the NOESY spectrum (Figure 3), NOE correlations of H3-26 (δH 1.00) with H-5 (δH 1.41)/H-6 (δH 2.04)/H-8ax/H-9 (δH 3.36) and H3-25 (δH 0.82) with H-7ax (δH 1.12) indicated that H-6, H-7ax, H-8ax, 9-OH, and H3-26 were axially oriented, while H-5, H-9, and H3-25 were equatorially oriented. The same procedure described for the synthesis of 1a was adopted for 2, furnishing the major bis-acetonide product 2a (Scheme 1). Similarly, in the 13C NMR spectrum of 2a (see Supporting Information), the chemical shifts of C-33 at δC 20.5 and C-32 at δC 30.1 further confirmed the relative configuration of C-19/C-20/C-21 bearing the contiguous hydroxy/methyl/ hydroxy groups was syn/syn.8 The NOE correlations observed for the polyol chain of 2a in the NOESY spectrum were almost the same as those of 1a (see Supporting Information), which further supported the assigned relative configuration of 2. The absolute configuration of 2 was also determined by the modified Mosher’s ester method. In the procedure, 2a was treated with either (R)- or (S)-MTPA-Cl to yield the 17-mono(S)- and 17-mono-(R)-MTPA esters 2b and 2c, respectively, and a standard Mosher’s ester analysis of the 1H NMR data collected for 2b and 2c (Figure 4) indicated that the absolute configuration of the polyol chain segment of 2 was also 17S, 19R, 20S, 21S. The molecular formula of nahuoic acid D (3) was determined as C32H54NaO7 by HRESIMS. The 1H and 13C NMR spectroscopic data of 3 (Table 1 and Table 2) closely resembled those of 2, with the only differences being the addition of one oxymethine (δH 3.78, δC 66.0, C-19) and one methylene (δH 1.31, δC 43.9, C-20) in 3. Cross-peaks observed between H-17 (δH 3.63) and H-16 (δH 2.05, 1.95)/H-18 (δH 1.31), between H-19 (δH 3.78) and H-18/H-20 (δH 1.43), and between H-20 and H-21 (δH 3.74) in the COSY spectrum (Figure 1) demonstrated that the long polyol chain was extended by the additional oxymethine (−OCH-19) and methylene (−CH2-20) groups. Additional COSY correlations of δH 4.53 with H-21 (δH 3.74), δH 4.40 with H-19, δH 4.34 with H-23 (δH 3.16), and δH 4.31 with H-17 led to the assignment of the four exchangeable protons, thereby accomplishing the establishment of the planar structure of 3. In a similar manner, the relative configuration of the C-17/C19/C-21/C-22/C-23 cluster located at the polyol chain was deduced as anti/syn/syn/syn by Kishi’s Universal NMR Database (Figure 2). The relative configuration of the decalin ring moiety was determined to be the same as that of 2 according to their identical NOE correlations (see Supporting

that of 1a. In the NOESY spectrum of 1a (Figure 3), a NOE correlation of H3-26 (δH 1.24) with H-5 (δH 1.63) established the decalin ring junction was cis.9 The coupling constant values of J5−6 (3.5 Hz), J6−7 (10.5 Hz), J7−8 (5.5 Hz), and J8−9 (almost zero) implied the ring encompassing C-5 to C-10 was in a chair conformation, and H-6, H-7, and OH-9 (δH 4.02) were in axial positions. The signal for H-4 with coupling constants to both H-5 and H-13 (J4−5 = 9.0 Hz, J4−13 = 8.5 Hz) demonstrated these protons were in axial positions of the six-membered ring composed of C-4, C-5, and C-10 to C-13. Furthermore, in the NOESY spectrum of 1a, NOE correlations from H3-26 to H-6 (δH 2.06)/H-9 (δH 4.02) indicated H-6/H-9/H3-26 to be on the same side with H-9 equatorial and H-6 and H3-26 axial, while NOE correlations from H3-25 (δH 0.94) to H-4 (δH 2.60)/H-7 (δH 4.13), from H-7 to H-4/H-8 (δH 4.44), and from H3-27 (δH 1.16) to H-13 (δH 1.79) suggested H-4/H-7/ H-8/H3-25/H3-27 was on the other side with H-8/H3-25/H327 equatorial and H-4/H-7/H-13 axial as shown. Moreover, the Δ2, 3 and Δ14, 15 trisubstituted olefins were assigned E configurations on the basis of NOE correlations of H-4 (δH 2.60) with H3-24 (δH 1.67) and of H-13 with H-15 (δH 4.96) (Figure 3). The absolute configuration of 1 was further determined by the modified Mosher’s ester method. Treatment of nahuoic acid B acetonide derivative (1a) with R-MTPA-Cl and SMTPA-Cl, under standard acylation conditions, afforded 17mono-S-MTPA-ester (1b) and 17-mono-R-MTPA-ester (1c), respectively (Scheme 1). The 1H chemical shifts around the C17 site of esterification were assigned by 1 H NMR spectroscopic analysis (see Supporting Information), and the analysis of ΔδS−R values allowed the absolute configuration of C-17 to be defined as S (Figure 4). Correspondingly, the absolute configuration of the C-17/C-19/C-20/C-21 cluster was established as 17S, 19R, 20S, and 21S. It was noted that the 9-OH group was not converted to the corresponding Mosher’s ester during the MTPA esterification process, possibly due to steric hindrance, which prevented the determination of the absolute configuration of the decalin unit by the Mosher’s ester method. The molecular formula of nahuoic acid C (2) was determined to be C30H50O6 by HRESIMS. The 1H and 13C NMR spectroscopic data of 2 (Table 1 and Table 2) were very similar to those of 5, with the only obvious difference of the addition of a shielded methylene and the absence of an oxymethine. Detailed analysis of 1D and 2D NMR spectra revealed that the planar structure of 2 was a deoxygenated analogue of 5 at C-7. Similarly, the relative configuration of the C-17/C-19/C-20/C-21 cluster in 2 was deduced as anti/syn/ syn by application of Kishi’s Universal NMR Database (Figure 2). The relative configuration of the decalin ring was E

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13R, 17S, 19R, 20S, 21S for 2, 4R, 5R, 6S, 9R, 10R, 13R, 17S, 19R, 21R, 22S, 23S for 3, and 4R, 5S, 6R, 7R, 9R, 10R, 13R, 17S, 19R, 21R, 22S, 23S for 4, respectively. The bioactivities of compounds 1−5 were evaluated. Unfortunately, none of the five compounds showed obvious inhibitory activity against acetylcholinesterase, nor did they exhibit any cytotoxicity toward the cancer cell lines H1975, K562, BGC 823, MCF-7, HL-60, and Huh-7.These compounds also failed to display antibacterial activity against Staphylococcus aureus and Shewanella oneidensis MR-1, as well as antibiofilm activity against S. aureus biofilm. Although 1−5 could inhibit the overall extent of biofilm formation of S. onedensis MR-1 to the levels of 63%, 94%, 95%, 91%, and 98%, respectively, at a concentration of 200 μg/mL, further bioassay results revealed that their MIC values were not less than 200 μg/mL, because their inhibition ratios had no dose-dependent effect with concentration. Although the study did not find potent bioactivities of 1−4, it revealed a series of analogues of the novel selective SETD8 inhibitor nahuoic acid A (5) for the first time.

Information) and coupling constants. In order to establish the absolute configuration of 3, multistep chemical derivatization experiments were performed. Compound 3 was subjected to a methyl esterification reaction followed by a reaction to form the acetonides as described above. Unfortunately, the yield of the bis-acetonide was low, and there were several bis-acetonide derivatives produced with no major product, which ultimately interrupted the subsequent Mosher’s esterification for the determination of the absolute configuration. The molecular formula of nahuoic acid E (4) was determined as C32H54NaO8 by HRESIMS. The 1H and 13C NMR spectroscopic data of 4 (Table 1 and Table 2) were highly similar to those of 3. Comparison of the NMR data for 3 and 4 suggested that the only difference was the C-7 methylene was oxygenated to be a methine (δC 66.3) in 4, which was confirmed by the COSY spectrum, showing correlations of H-7 (δH 3.53) with H-6 (δH 1.91)/H-8 (δH 1.88, 1.63) (see Supporting Information). Thus, the planar structure of 4 was established. In a similar way, the relative configuration of the polyol side-chain was readily deduced as anti/syn/syn/syn by Kishi’s Universal NMR Database (Figure 2), and the relative configuration of the decalin ring moiety was determined to be identical with that of 5 by their accordant NOE correlations (see Supporting Information). We also failed to establish the absolute configuration of 4 by multistep chemical derivatization experiments as described above for 3. For compounds 2−4, the geometries of the two double bonds Δ2,3 and Δ14,15 were also assigned E configurations based on the NOE correlations of H-13 with H-15 and of H-4 with H-24 in their NOESY spectra. Although the relative configurations between the decalin system and the side-chain in 1−4 were not established by the data presented above, the absolute configurations of 1−4 could be determined by comparison of their ECD spectra with that of 5. The complete relative configuration and the absolute configuration (4R, 5S, 6R, 7R, 9R, 10R, 13R, 17S, 19R, 20S, 21S) of 5 were determined by modified Mosher’s ester analysis, and its absolute configuration was confirmed by the consistency of its calculated ECD and experimental ECD spectra.5 The experimental ECD spectra of 1−5 were almost the same (Figure 5), and the relative configurations of chiral carbons in the decalin ring unit and the C-19/C-20/C-21 cluster of compounds 1−5 were correspondingly consistent, respectively. Thus, it was reasonable to assign the absolute configuration of 4R, 5S, 6R, 7S, 8S, 9S, 10R, 13R, 17S, 19R, 20S, 21S for 1, 4R, 5R, 6S, 9R, 10R,

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a MCP 500 polarimeter (Anton Paar). UV spectra were recorded using a UV-2600 spectrophotometer (Shimadzu). ECD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd.). IR spectra were determined with an FT-IRNICOLET spectrophotometer. 1H and 13 C NMR and 2D NMR spectra were acquired with a Bruker AV-500 MHz NMR spectrometer with TMS as reference. ESIMS and HRESIMS spectroscopic data were acquired with an amaZon SL ion trap mass spectrometer and MaXis quadrupole-time-of-flight mass spectrometer (Bruker), respectively. Semipreparative reversed-phase (SP-RP) HPLC was performed on a Shimadzu LC-20A preparative liquid chromatography system with a YMC-Pack ODS column, 250 × 20 mm i.d., S-5 μm. RP-MPLC (reversed-phase-medium pressure preparative liquid chromatography) was carried out using the CHEETAH MP200 system (Agela Technologies) and Claricep Flash columns filled with ODS (40−63 μm, YMC). Sephadex LH-20 (GE Healthcare) was used for the column chromatographic (CC) column. Silica gel (200−300 mesh) for CC and GF254 for TLC were obtained from Yantai Jiangyou Silica Gel Development Co., Ltd. Optical density (OD) measurements were determined on an Enspire multimode microplate reader (Varioskan Flash). Biological Material. Strain SCSGAA0027 was isolated from the South China Sea (18°11′ N, 109°25′ E) gorgonian coral Melitodes squamata. The strain (GenBank accession JN049466) was identified as a Streptomyces sp. based on NCBI BLAST analysis of its partial 16S rRNA sequence, which is 99% identical with that of the strain Streptomyces f radiae NBRC 12773 (NR112270). Strain SCSGAA0027 was deposited in the RNAM center, South China Sea Institute of Oceanology, Chinese Academy of Sciences. Fermentation and Extraction. Strain Streptomyces sp. SCSGAA0027 was cultured in 2500 replicate 500 mL Erlenmeyer flasks each containing 120 mL of modified ISP2 fermentation medium (yeast extract 0.4%, malt extract 0.5%, glucose 0.4%, artificial sea salt 1.5%, vitamin B1 0.01%, vitamin B6 0.01%, vitamin lactoflavin 0.01%, nicotinic acid 0.01%, biotin 0.01%, phenylalanine 0.01%, alanine 0.03%, pH 7.2−7.4). These cultures were grown on a rotary shaker (200 rpm) at 28 °C for 5 days. At the end of the fermentation period, the culture broth was separated from the mycelium by filtration. The mycelium was extracted with acetone, while the culture filtrate was extracted with EtOAc. A combined organic extract was afforded. Isolation and Purification. The total extract (50 g) was adsorbed on silica gel (YMC-gel ODS-A, 30 g) and subjected to RP-MPLC, eluting with a step gradient of MeOH/H2O solvent mixtures (increasing the MeOH by 20% per 3000 mL from 30% to 100%

Figure 5. Comparison of experimental ECD spectra of 1−5. F

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MeOH, 40 mL/min flow rate). The 70% MeOH/H2O fraction (5 g) was further fractionated by RP-MPLC with a MeCN/H2O gradient solvent system (from 30% to 60% acetonitrile, 20 mL/min flow rate) to obtain four subfractions (F-1, F-2, F-3, F-4). Fraction F-1 was further purified by repeated SP-RP-HPLC (YMC-Pack ODS column, 250 × 20 mm i.d., S-5 μm; 5 mL/min flow rate) with MeCN/H2O (41:59) to obtain 1 (10 mg, tR = 23 min), 4 (5 mg, tR = 27 min), and 5 (20 mg, tR = 31 min). Compounds 2 (8 mg, tR = 42 min) and 3 (5 mg, tR = 52 min) were obtained from fraction F-3 by repeated SP-RPHPLC with MeCN/H2O (55:45). Nahuoic acid B (1): pale yellow oil; [α]25D −5.4 (c 0.82, MeOH); UV (MeOH) λmax (log ε) 197 (4.26) nm; ECD (0.49 mM, MeOH) λmax (Δε) 226 (+3.18) nm; IR (KBr) νmax 3411, 2963, 2936, 2876, 1686, 1643, 1452, 1386, 1275, 1231 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 561.3393 [M + Na]+ (calcd for C30H50NaO8, 561.3398). Nahuoic acid C (2): pale yellow oil; [α]25D −1.8 (c 0.98, MeOH); UV (MeOH) λmax (log ε) 197 (4.24) nm; ECD (0.66 mM, MeOH) λmax (Δε) 226 (+3.37) nm; IR (KBr) νmax 3417, 2934, 2874, 2652, 1686, 1643, 1460, 1384, 1273, 1237 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 529.3506 [M + Na]+ (calcd for C30H50NaO6, 529.3500). Nahuoic acid D (3): pale yellow oil; [α]25D −1.3 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 208 (4.39) nm; ECD (0.65 mM, MeOH) λmax (Δε) 226 (+2.05) nm; IR (KBr) νmax 3411, 2961, 2876, 1686, 1645, 1460, 1384, 1275, 1234 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 573.3763 [M + Na]+ (calcd for C32H54NaO7, 573.3762). Nahuoic acid E (4): pale yellow oil; [α]25D −2.6 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 197 (4.28) nm; ECD (0.61 mM, MeOH) λmax (Δε) 227 (+2.51) nm; IR (KBr) νmax 3408, 2961, 2933, 2874, 1686, 1645, 1460, 1384, 1275, 1234 cm−1; 1H and 13C NMR data, Table 1 and Table 2; HRESIMS m/z 589.3721 [M + Na]+ (calcd for C32H54NaO8, 589.3711). Preparation of Bis-acetonide 1a. Nahuoic acid B (1, 8 mg) was treated with 1% sulfuric acid in MeOH (5 mL) and incubated for 5 h at 70 °C. Consequently, the reaction mixture was quenched with purified H2O and the aqueous phase was extracted twice with CH2Cl2. Afterward, the CH2Cl2 solvent was removed under reduced pressure to obtain a residue. Further, the residue (6 mg) was dissolved in 2,2dimethoxypropane (2 mL) and MeOH (1 mL), and pyridinium-ptoluenesulfonate (5 mg) was added. The reaction was allowed to stir for 24 h at room temperature (rt). At the end of the reaction, the reaction mixture was quenched with 2 mL of 5% aqueous NaHCO3; then the aqueous phase was extracted twice with CH2Cl2. The CH2Cl2 solvent was removed under reduced pressure, and the residue was purified by C-18 RP-HPLC (91:9 MeCN/H2O) to provide compound 1a (2 mg, tR = 48 min). For 1H and 13C NMR data for 1a, see the Supporting Information. HRESIMS m/z 659.4481 [M + Na]+ (calcd for C37H64NaO8, 659.4493). Preparation of Mosher’s Esters 1b and 1c. Compound 1a (1 mg) was dissolved in 600 μL of pyridine, and dimethylaminopyridine (1 mg) and (S)-MTPACl (20 μL) were then added in sequence. The reaction mixture was stirred at rt for 24 h and afterward quenched with two drops of water. The solution was purified by C-18 RP-HPLC (95:5 MeCN/H2O) to obtain a mono-(R)-MTPA ester derivative, 1b (1 mg, tR = 66 min). At the same time, another portion of 1a (1 mg) was treated with 600 μL of pyridine, dimethylaminopyridine (1 mg), and (R)-MTPACl (20 μL). The reaction was performed at the same conditions as above, and the major product was purified by C-18 RPHPLC (98:2 MeCN/H2O) to yield the mono-(S)-MTPA ester derivative 1c (1 mg, tR = 52 min). 1b: 1H NMR (CDCl3, 500 MHz) δH 6.472 (1H, d, J = 10.0 Hz, H-3), 5.200 (1H, br s, H-17), 4.842 (1H, brs, H-15), 4.410 (1H, d, J = 5.5 Hz, H-8), 4.109 (1H, dd, J = 5.5, 9.5 Hz, H-7), 4.001 (1H, br s, H-9), 3.738 (1H, d, J = 10.5 Hz, H-19), 3.182 (1H, d, J = 9.5 Hz, H-21), 2.531 (1H, ddd, J = 8.0, 9.5, 10.0 Hz, H-4), 2.389 (1H, m, H-16a), 2.202 (1H, m, H-16b), 1.719 (1H, d, J = 9.5 Hz, H-13), 1.620 (3H, s, H-28), 1.232 (3H, s, Me-26), 1.106 (3H, s, Me-27), 0.921 (3H, d, J = 6.5 Hz, Me-25), 0.918 (3H, d, J = 6.5 Hz, Me-23), 0.785 (3H, d, J = 7.0 Hz, Me-30), 0.770 (3H, d, J = 7.0 Hz,

Me-29); HRESIMS m/z 875.4851 [M + Na] + (calcd for C47H71F3NaO10, 875.4892). 1c: 1H NMR (CDCl3, 500 MHz) δH 6.491 (1H, J = 10.5 Hz, H-3), 5.188 (1H, br s, H-17), 4.414 (1H, d, J = 5.0 Hz, H-8), 4.116 (1H, dd, J = 5.5, 10.0 Hz, H-7), 4.003 (1H, br s, H-9), 3.635 (1H, d, J = 10.0 Hz, H-19), 3.141 (1H, d, J = 9.0 Hz, H21), 2.550 (1H, ddd, J = 8.0, 9.5, 10.0 Hz, H-4), 1.758 (1H, d, J = 9.5 Hz, H-13), 1.640 (3H, s, H-28), 1.208 (3H, s, Me-26), 1.115 (3H, s, Me-27), 0.926 (3H, d, J = 6.0 Hz, Me-25), 0.914 (3H, d, J = 6.0 Hz, Me-23), 0.749 (3H, d, J = 6.5 Hz, Me-30), 0.729 (3H, d, J = 7.0 Hz, Me-29); HRESIMS m/z 875.4834 [M + Na] + (calcd for C47H71F3NaO10, 875.4892). Preparation of Bis-acetonide 2a. Nahuoic acid B (2, 6 mg) was derivatized to afford the major bis-acetonide 2a residue as described above. Subsequently, the residue was purified by C-18 RP-HPLC (95:5 MeCN/H2O) to provide compound 2a (1.2 mg, tR = 48 min). For 1H and 13C NMR data for 2a, see the Supporting Information. HRESIMS m/z 583.3977 [M − H2O + Na]+ (calcd for C34H56NaO6, 583.3969). Preparation of Mosher’s Esters 2b and 2c. Compound 2a (0.6 mg) was esterified with (S)-MTPACl (20 μL) as previously described. The resulting solution was purified by C-18 RP-HPLC (97:3 MeCN/ H2O) to obtain the mono-(R)-MTPA ester derivative 2b (0.5 mg, tR = 66 min). Another portion of 2a (0.6 mg) was esterified with (S)MTPACl (20 μL) as described above, and the resulting solution was purified by C-18 RP-HPLC (95:5 MeCN/H2O) to yield the mono(S)-MTPA ester derivative 2c (0.5 mg, tR = 60 min). 2b: 1H NMR (CDCl3, 500 MHz) δH 6.541 (1H, d, J = 10.0 Hz, H-3), 5.208 (1H, br s, H-17), 4.936 (1H, brs, H-15), 3.750 (1H, d, J = 10.0 Hz, H-19), 3.627 (1H, br s, H-9), 3.187 (1H, d, J = 9.0 Hz, H-21), 2.734 (1H, ddd, J = 8.0, 9.5, 10.0 Hz, H-4), 2.425 (1H, m, H-16a), 2.221 (1H, m, H-16b), 1.787 (1H, d, J = 9.5 Hz, H-13), 1.752 (3H, s, Me-28), 1.341 (3H, s, Me-33), 1.258 (3H, s, Me-32), 1.107 (3H, s, Me-27), 1.068 (3H, s, Me-26), 0.921 (3H, d, J = 6.5 Hz, Me-23), 0.782 (3H, d, J = 6.0 Hz, Me-30), 0.770 (3H, d, J = 6.5 Hz, Me-29), 0.728 (3H, d, J = 7.0 Hz, Me-25); HRESIMS m/z 799.4380 [M − H2O + Na]+ (calcd for C44H63F3NaO8, 799.4367). 2c: 1H NMR (CDCl3, 500 MHz) δH 6.562 (1H, d, J = 10.0 Hz, H-3), 5.198 (1H, br m, H-17), 4.992 (1H, br s, H15), 3.635 (1H, br s, H-9), 3.632 (1H, d, J = 10.0 Hz, H-19), 3.137 (1H, d, J = 10.5 Hz, H-21), 2.738 (1H, ddd, J = 8.0, 9.5, 10.0 Hz, H-4), 2.436 (1H, m, H-16a), 2.311 (1H, m, H-16b), 1.830 (1H, d, J = 9.5 Hz, H-13), 1.782 (3H, s, Me-28), 1.328 (3H, s, Me-33), 1.269 (3H, s, Me-32), 1.118 (3H, s, Me-27), 1.070 (3H, s, Me-26), 0.913 (3H, d, J = 7.0 Hz, Me-23), 0.744 (3H, d, J = 7.0 Hz, Me-30), 0.737 (3H, d, J = 7.0 Hz, Me-29), 0.726 (3H, J = 7.0 Hz, Me-25); HRESIMS m/z 799.4389 [M − H2O + Na]+ (calcd for C44H63F3NaO8, 799.4367). In Vitro Antibiofilm Assay. Biofilm cultivation in sterile 96-well polystyrene microliter plates was carried out essentially as previously described.10,11 Briefly, overnight cultures of two strains grown in LB medium were adjusted to an optical density at 600 nm of 0.05 in LB medium. The diluted cultures with different concentrations of compounds were transferred to the wells of polystyrene microliter plates (150 μL per well) and incubated for the desired time. Afterward, the wells were washed with distilled H2O, dried in an inverted position, and stained with 170 μL of crystal violet solution (0.5% w/v) for 30 min. The wells were then washed three times with distilled H2O, dried in an inverted position, and dissolved in 200 μL of sodium acetate solution (33% w/v), and the absorbance was determined at 560 nm. The experiment was run in three replicates.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00805. NMR data of 1a and 2a, 1D/2D NMR and HRESIMS spectra of 1, 1a, 2, 2a, 3, and 4 (PDF) G

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-8902-2112. Fax: +81-20-8445-8964. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support provided by the Regional Innovation Demonstration Project of Guangdong Province Marine Economic Development (GD2012-D01-002 and GD2012-D01-001), Natural Science Foundation of China (41376160), Natural Science Foundation of Guangdong Province, China (2015A030310349), National Marine Public Welfare Research Project of China (201305017), Strategic Leading Special Science and Technology Program of Chinese Academy of Sciences (XDA100304002), National High Technology Research and Development Program of China (863 Program, 2012AA092104), and Marine Fishery Science and Technology Promotion Project of Guangdong Province (A201301B05).

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